Project Summary Exosomes are from the intraluminal vesicles (ILV) of multivesicular bodies (MVBs) produced via endocytic process. Mature MVBs can fuse with lysosomes to deliver their contents for degradation and recycling or fuse with the plasma membrane where ILVs are released as exosomes. Recent studies have indicated that enhanced exosome excretion in arterial smooth muscle cells (SMCs) is an essential mechanism triggering or promoting calcifying nidus formation and extracellular matrix (ECM) mineralization in the arterial wall under different pathological conditions. However, little is known so far how MVB fate and exosome excretion are controlled by lysosomes in SMCs and whether lysosomal dysfunction increases exosome excretion from SMCs to activate or accelerate vascular calcification process. The present grant proposal will test a central hypothesis that lysosomal acid ceramidase (AC)-mediated sphingolipid metabolism plays a crucial role in the control of lysosome trafficking or fusion to MVBs and subsequent exosome excretion, maintaining the normal phenotype and function of SMCs. AC gene defect or functional deficiency may disturb lysosome degradation of MVBs, increasing exosome excretion and resulting in calcifying nidus formation and ultimate vascular calcification under pathological conditions. To test this hypothesis, three Specific Aims are proposed. Specific Aim 1 will determine whether exosome excretion in arterial SMCs is fine controlled by lysosomal AC activity and whether the deficiency of this AC regulation causes arterial calcification in smooth muscle-specific AC gene knockout mice (Asah1fl/fl/SMcre) with analysis of exosomes, SMC phenotypes, and calcification in the arterial wall and cultured coronary arterial SMCs. Specific Aim 2 attempts to test whether lysosomal AC- mediated sphingolipid signaling regulates lysosome trafficking to and fusion with MVBs to limit exosome excretion from SMCs with gene deletion (Asah1fl/fl/SMcre), CRISPR-Cas9 gene editing, and SM22? promoter- driven gene rescuing. In Specific Aim 3, we will address whether the AC regulation of lysosome trafficking or fusion to MVBs is attributed to its action on lysosomal TRPML1 channel activity and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging with GCaMP3-ML as an indicator. To our knowledge, these proposed studies will represent the first effort to investigate the lysosome regulation of exosome excretion from SMCs and associated pathogenic role in vascular calcification. The findings will provide new insights into the pathogenesis of arterial calcification and identify lysosomal AC as a therapeutic target for prevention or treatment of vascular calcification.